BACKGROUND: Carnitine palmitoyltransferase 1 (CPT1) is the rate-limiting enzyme governing the entry of long-chain acyl-CoAs into mitochondria. Treatments with CPT1 inhibitors protect against insulin resistance in short-term preclinical animal studies. We recently reported that mice with muscle isoform CPT1b deficiency demonstrated improved insulin sensitivity when fed a High Fat-Diet (HFD) for up to 5 months. In this follow up study, we further investigated whether the insulin sensitizing effects of partial CPT1b deficiency could be maintained under a prolonged HFD feeding condition. METHODS: We investigated the effects of CPT1b deficiency on HFD-induced insulin resistance using heterozygous CPT1b deficient (Cpt1b+/-) mice compared with Wild Type (WT) mice fed a HFD for a prolonged period of time (7 months). We assessed insulin sensitivity using hyperinsulinemic-euglycemic clamps. We also examined body composition, skeletal muscle lipid profile, and changes in the insulin signaling pathways of skeletal muscle, liver, and adipose tissue. RESULTS: We found that Cpt1b+/- mice became severely insulin resistant after 7 months of HFD feeding. Cpt1b+/- mice exhibited a substantially reduced glucose infusion rate and skeletal muscle glucose uptake. While Cpt1b+/- mice maintained a slower weight gain with less fat mass than WT mice, accumulation of lipid intermediates became evident in the muscle of Cpt1b+/- but not WT mice after 7 months of HFD feeding. Insulin signaling was impaired in the Cpt1b+/- as compared to the WT muscles. CONCLUSION: Partial CPT1b deficiency, mimicking CPT1b inhibition, may lead to impaired insulin signaling and insulin sensitivity under a prolonged HFD feeding condition. Therefore, further studies on the potential detrimental effects of prolonged therapy with CPT1 inhibition are necessary in the development of this potential therapeutic strategy.